The hepatitis C virus (HCV) causes several liver diseases, including liver cancer. The HCV genome is a plus-stranded RNA that encodes the single polyprotein processed into at least 10 mature polypeptides.
The structural proteins are located in the amino terminal quarter of the polyprotein, and the non-structural (NS) polypeptides in the remainder (for a review, see HOUGHTON, 1996). The genome organisation resembles that of flaviviruses and pestiviruses and HCV is now considered to be a member of the flaviviridae family.
The gene products of HCV are, from the N-terminus to the C-terminus: core (p22), E1 (gp35), E2 (gp70), NS2(p21), NS3 (p70), NS4a (p4), NS4b(p27), NS5a (p58), NS5b (p66), as disclosed in FIG. 1. Core, E1and E2 are the structural proteins of the virus processed by the host signal peptidase(s). The core protein and the genomic RNA constitute the internal viral core and E1 and E2 together with lipid membrane constitute the viral envelop (DUBUISSON et al., 1994; GRAKOUI et al., 1993; HIGIKATA et al., 1993.).
The NS proteins are processed by the viral protein NS3 which has two functional domains: one (Cro-1), encompassing the NS2 region and the N-terminal portion of NS3, which cleaves autocatalytically between NS2 and NS3, and the other (Cro-2), located solely in the N-terminal portion of NS3, cleaves the other sites downstream NS3 (BARTENSCHLAGER et al; 1995; HIGIKATA et al;, 1993).
Various HCV protein-protein interactions have already been identified, notably by two hybrid methods. Noticeably, FLAJOLET et al; (2000) have shown interactions between NS3 and NS4A proteins as well as between NS4A and NS2 proteins. These authors have also shown core-core, NS3-E2, NS5A-E1, NS4A-NS3 and NS4A-NS2 interactions. Covalent as well as non-covalent interactions between E1 and E2 have been shown by PATEL et al; (1999). The protein interactions between NS3 and the HCV RNA helicase have also been described (MIN et al; 1999; GALLINARI et al., 1999) as well as interaction between NS3 and NS4A (URBANI et al., 1999; DI MARCO et al., 2000; BUTKIEWICZ et al., 2000).
However, the prior art methods allow the determination of interactions between full length proteins or large domains of proteins encoded by the genome of the hepatitis C virus which may contain more than one region of interaction with one or several HCV proteins. BUTKIEWICZ et al. (2000) discloses the interaction between the NS3 protease and a small peptide derived from NS4A. However, BUTKIEWICZ et al. (2000) discloses exclusively in vitro assays for interactions between the small peptides derived from NS4A and the NS3 protease from HCV which may not be of physiological relevance.
There is a need in the art for polypeptides that contain the minimal aminoacid sequence that is able to bind specifically with a naturally-occurring HCV protein in physiological conditions in order to design new tools for therapeutic and detection purposes related to HCV.